1. Field of the Invention
The present invention relates to apparatus and methods for making fluorescence measurements and sorting large multi-cellular organisms in a flowing liquid.
2. Background
WatchFrog (Paris, France) has developed a technique for sensitively testing for pollutants in the environment and for pharmaceutical testing. Xenopus tadpoles “light up” (exhibit fluorescence) in response to a pollutant (or drug), and can indicate the presence of several chemical species at the same time. This is described in publicly available literature (for example, Turque et al. 2005) and in US Patent Application 20060101528 (Demeneix and Turque) on transgenic Xenopus, and is summarized below:
The basic principle involves creating genetic constructions that enable a GFP (Green Fluorescent Protein) to be expressed in response to the physiological action of whatever type of molecules a customer may be interested in. This ‘molecular dosimeter’ is then incorporated in a Xenopus larva, thereby taking into account all the biochemical regulations that can respond in vivo to the sample being tested.
For example, if an endocrine disrupter is present, it will activate the response element of various hormones, such as estrogen or thyroid hormone, triggering the synthesis of fluorescent proteins. The fluorescence is visible through the transparency of the organism, and can therefore be detected and quantified without sacrificing the animal. The larvae simply need to be placed in the liquid sample to implement the test. The genetic constructions can be altered as required to produce a tailor-made range of tests to respond to various disruptive or pharmacological effects.
This test methodology combines the advantages of in vivo with the flexibility of in vitro. It rapidly and simply furnishes accurate information of high sensitivity and specificity, together with low cost, economic use of material, and the potential for automation.
Xenopus (the choice of species) has a complete immune system, as well as a more complex heart and circulatory system. In addition, in terms of endocrine physiology, the conservation of biochemical mechanisms between Xenopus and humans has been demonstrated and proved. Xenopus is an investigated and recognized model in the research world.
In addition, Xenopus allows a number of pharmaceutical applications. For example: Xenopus is again relevant in that it very rapidly develops a vascular system and a complex central nervous system in the course of its growth. Thus we are able to develop target-models to test new molecules of angiogenic or neurological interest.
Also known in the art are various methods of detecting particles. For example, U.S. Pat. No. 6,765,656 to the present inventor teaches a fountain flow cytometer, wherein a sample of fluorescently tagged cells flows up a tube toward a digital CCD or CMOS camera and fore-optics. See FIG. 1 (Prior Art). The cells are illuminated in the focal plane by a laser through a transparent end element. When the cell(s) pass through the digital camera focal plane, they are imaged by the camera and a lens assembly, through a transparent window and a filter that isolates the wavelength of fluorescent emission. The fluid in which the cells are suspended then passes by the window ad out the drain tube.
FIG. 1 (Prior Art) shows a schematic diagram of the epifluorescent Fountain Flow™ Cytometer 100 as used in this study. A Sample 102 of fluorescent organisms flows through the flow cell 104 toward the digital camera 106 and fore-optics 108. The cells are illuminated in the focal plane 110 by a laser 112. Then the cell(s) pass through the CCD camera focal plane and they are imaged by the CCD camera and lens assembly through the transparent flow cell window, using a filter 114 that isolates the wavelength of fluorescence emission. The fluid in which the cells are suspended then passes by the window 118 and effluent 120 flows out the flow cell drain tube 116 (in the path indicated by the arrows).
A flow block may be used as flow cell 104, as shown in FIG. 2 (Prior Art) wherein the sample 102 enters the flow block through entrance tube 202 via input tubing 208, is forced up and under an imaging window 118, and flows back down to exit through drain tube 116 and out effluent exit tubing 206.
FIG. 2 (Prior Art) shows a schematic drawing of an aluminum flow block used as flow cell 104 with the device in FIG. 1. The sample 102 enters the flow block 104 through a flexible tubing (Tygon™ or the like) input tubing 208 connected to a stainless steel entrance tube 202 and exists through stainless steel drain tube 116 to effluent exit tubing 206. Two vertical 8-mm holes have been drilled into the aluminum flow block: an entrance hole 210 and an exit hole 214. As the sample flows up the internal entrance hole 210, it passes through the focal plane 110 of the digital (e.g. CCD or CMOS) camera 106. This hole 210 is generally painted black to reduce scattered light. A Teflon tape gasket 216 is sandwiched between the aluminum flow block and a circular BK7 window 118, and tightly held with a screw-on brass cap 218. The gasket is cut to allow the sample to be viewed through window 118. Sample 102 then passes down exit hole 214 to drain tube 116. LED illumination may be used as shown in FIG. 3 (Prior Art).
FIG. 3 (Prior Art) shows a schematic diagram of an LED-illuminated epifluorescent Fountain Flow Cytometer 300. A sample of fluorescently tagged cells flows through the flow cell 104 toward the digital camera 106 and fore-optics 108. The cells are illuminated in the focal plane by an LED 302. When the cell(s) pass through the CMOS camera focal plane 110, they are imaged by the camera and lens assembly 108 through the transparent flow cell window 118, and a filter (not shown) that isolates the wavelength of fluorescence emission. The fluid in which the cells are suspended then passes by the window 118 and out the flow cell drain tube 116. (Note: in the current embodiment a peristaltic pump is not used.)